The zebrafish is an excellent experimental system for the study of embryonic patterning, organ function, and disease. There are over 2000 independent mutants with specific developmental defects, and a major goal of the community is to define the affected genes. Mutations can easily be mapped onto chromosomes of the zebrafish genome; however, low marker density precludes positional cloning of most genes. We have successfully utilized an amplified fragment length polymorphism (AFLP) technique to define genetic markers less than 0.3 cM from mutations leading to defective hematopoiesis. The availability of close markers has allowed chromosomal walking to several genes. Despite this success, each positional cloning project requires a separate AFLP marker screen. To further define the zebrafish genome a genetic map of 20,000 AFLP markers will be created using a panel of DNAs from heat shock-derived isogenic adult fish. This project will define genetic markers roughly every 0.3 cM, ensuring a close marker for positional cloning of any gene. This map will be assembled within one year and will complement the microsatellite markers being derived by other laboratories which require more time to generate and are costly. To further improve marker density and to provide a potential candidate gene approach to the zebrafish system, we have created and characterized four zebrafish radiation hybrid panels. For the best characterized panel, we have demonstrated a retention frequency of 24 percent and established two linkage groups of 25 markers on one zebrafish chromosome. Base on the evaluation of markers derived from several chromosomal walks, the panel can resolve physical distances of 150 kB, ideal for position cloning projects. The order of genetic markers in the chromosomal walk was the same as that determined by the radiation hybrid panel mapping. The additional three radiation hybrid panels are being similarly evaluated. To correlate the radiation hybrid map with the genetic map, we plan to type 1000 known genetic markers (microsatellites and cDNAs) on the panel with the best resolution. 500 expressed sequence tags (ESTs) derived from cDNA libraries of various tissues and developmental stages will be mapped using this panel. This project will provide ample candidate genes for mutations and will further characterize synteny of the zebrafish and human genomes. The increased density of markers will enable investigators to more rapidly clone functionally important genes in the zebrafish, ensuring the success of this model system for understanding embryonic development, organ function, and disease.